In our long-standing collaboration with the laboratory of Dr. Lawrence Samelson (NCI) on the study of multi-protein interactions in signaling particles after T-cell activation we have succeeded to characterize the reversible formation of a four-molecular complex by sedimentation velocity analytical ultracentrifugation and isothermal titration calorimetry. A picture emerged where a circular arrangement of binary interactions adds binding specificity for the formation of the complex, overcoming promiscuity of modular binding interaces, while significant unfavorable entropy prevents the complex from having excessive stability. This interplay of specificity and enthalpy-entropy compensation fits well the proposed role of unstructured protein domains in signal transduction. We have continued our collaboration with Dr. Dan Sackett (NICHD) on the study of tubulin dimer dissociation. Tubulin self-assembles into microtubules, which are critical for the cellular homeostasis and for cell division, and therefore tubulin has long been a major target for cancer drugs. The high affinity of the tubulin dimer requires binding experiments to be carried out at very low concentration, which can be achieved by fluorescence-detected sedimentation velocity. Strikingly, we observed that the strength of dimerization is different for tubulin molecules from different species, but the dimerization interface is sufficiently conserved to allow cross-species dimerization. A major focus of our experimental work in the reporting period was the study of HIV-1 Gag assembly into virus-like particles, in collaboration with Dr. Alan Rein (NCI). This study exploits our new fluorescence detection capability for binding studies in sedimentation velocity analytical ultracentrifugation, and our advances in combining structural information gleaned from quenching with hydrodynamic resolution of different complexes in sedimentation velocity. We have observed a previously unknown dimerization interface most likely located in the nucleic acid binding domain of Gag. The interface is allosterically activated through nucleic acid binding, and involves two distinct conformational changes. We have identified involvement of the capsid domain and the matrix domain of Gag in this dimerization process, which may help targeting viral assembly to the cell membrane. This self-association mode may also be involved in binding specificity to package viral RNA into the particles. The newly discovered allosterically controlled interface may offer a target for inhibition of viral assembly. A different set of projects is exploiting our new sedimentation velocity methodology for studying highly concentrated solutions. The ability to measure macromolecular size-distribution and to quantify weak transient interactions in concentrated solutions allowed us to revisit the question of the solution state of eye lens crystallin, in collaboration with the laboratory of Dr. Graeme Wistow (NEI). The self-association state of crystallins at the extremely high concentrations in the eye lens is critical for understanding mechanism of cataract worldwide the leading cause for blindness. These experiments are ongoing. In parallel, we studied the behavior of therapeutic proteins at the high concentrations in formulation conditions, in collaboration with Dr. Kang Chen (FDA). Here, the goal is to establish protocols for the reliable measurement of immunogenic trace aggregates in therapeutic formulations to ensure drug safety. Other collaborative applications of AUC include studies of coated gold nanoparticles and their interactions with different proteins with Dr. Alioscka Souza (University of Sao Paolo), and the study of critical protein interactions in the hair cell signal transduction apparatus with the laboratory of Dr. Eric Gouaux (Vollum Institute and HHMI Oregon Health & Science University).